Illumination of diffusely scattering media

ABSTRACT

The invention provides a technique for increasing the illumination intensity of probe light in a diffusely scattering sample without increasing the power of the probe beam. Generally, an optical filter is used which permits a collimated probe beam of light to pass through to the sample, but which reflects back towards the sample much of the backscattered scattered probe light emerging at a wider range of angles. In particular embodiments a collimated laser beam is delivered to the sample through a multi-layer dielectric filter covering a portion of the sample. The filter is transmissive to the laser light at normal incidence, but reflective at shallower angles of incidence characteristic of the backscattered light.

FIELD OF THE INVENTION

The invention relates generally to the illumination of diffuselyscattering samples. In particular, the invention relates to techniquesfor reducing loss of incident light from within a sample, so thatintensity of the incident light within the sample is increased. Theinvention may, for example, be applied to spectroscopy to increase theintensity of spectral features to be detected.

INTRODUCTION

Various analytical applications involve the spectroscopic analysis ofdiffusely scattering media. Examples include the probing of livingtissue to determine tissue parameters such as bone composition asdiscussed in WO2006/61565, breast tissue, or blood glucose composition.The spectroscopic analysis of pharmaceutical tablets may be used todetermine crystalline states or purity on a production line, in postproduction testing, and when screening for counterfeits. Otherapplications include the laboratory analysis of a wide variety ofpowdered samples, turbid fluids, translucent materials and so forth.

Raman spectroscopy, in which incident light is shifted in wavelength byinelastic scattering within a medium, is frequently used in suchapplications because of its high degree of chemical specificity,although infrared absorption and emission spectroscopy are also widelyused. The cross section for Raman scattering is, however, particularlysmall, and obtaining a sufficiently high signal to noise ratio forspectral features of interest is challenging, especially in applicationsoutside of the laboratory in which less sensitive equipment may be used.In many practical applications, incident light intensity must belimited, for example to avoid damage to living tissue, and exposuretimes may also be limited, for example on a production line, or where ameasurement must be taken from a human patient in a reasonably shortlength of time. Of course, these and similar constraints also apply inthe case of various types of infrared and other spectroscopictechniques.

Consequently, it is generally desirable to maximise the spectral signalobtained using a particular intensity or power of incident light, whileminimising exposure times.

In other applications, it may be desirable to increase retention ofincident light within a scattering sample for other reasons, such as toincrease the rate of a chemical reaction triggered by the light, or toincrease the amount of incident light escaping from the sample in thearea where the light is introduced.

SUMMARY OF THE INVENTION

The invention seeks to address the above and other problems of therelated prior art.

Various spectroscopic techniques and other applications require thedirecting of a beam of incident light into a sample. In someapplications it is also desired to collect light scattered back out ofthe volume or from a surface of the sample, for example to detectspectral features in the collected light. Typically, the intensity ofincident light at the surface or within the volume of a diffuselyscattering sample, and hence the intensity of incident light scatteredback out of the sample is greatest close to the point of application ofthe incident light.

The invention provides a method of increasing the illumination of adiffusely scattering sample by a beam of incident light, such as a laserbeam or other substantially monochromatic beam of light, by covering aregion of the sample with a delivery filter, and directing the beam tothe sample through the filter. The filter has characteristics such thatthe light at the incident light wavelength which is diffusely scatteredback from the sample to the filter at a wider range of angles ofincidence than the incident beam is preferentially reflected back to thesample. Effectively, the filter acts as a unidirectional mirror,preventing loss of incident wavelength light, especially at the criticalpoint of application to the sample of the incident light beam whereintensities are greatest.

Some optical filter types, such as multi-layer dielectric filters havetransmission and reflection characteristics which shift in wavelength,typically to shorter wavelengths, with increasing angle of incidence.The delivery filter may therefore be provided, for example, by using amulti-layer dielectric filter having a transmission region which matchesthe wavelength of the incident light at the angle of incidence of thebeam, but which shifts away from the wavelength of the incident light atother angles of incidence. In this way, the incident beam, which iscollimated or semi-collimated to a small range of angles of incidencepasses through the filter into the sample, but the majority of diffuselyreflected light, which returns at a range of angles significantly widerthan the range of angles of incidence of the original beam, is reflectedback towards the sample, with only a small fraction passing away fromthe sample through the filter.

An example delivery filter is a narrow band pass filter with a band passregion matching the incident light wavelength, to transmit the incidentbeam at normal incidence, but which increasingly reflects light of thesame wavelength at larger angles of incidence. The same effect can beachieved using a notch filter or short wavelength transmission edgefilter, having a reflection or low transmission region which lies justabove the incident wavelength for normal incidence, but which shifts tocover the incident wavelength at larger angles of incidence.

According to one particular aspect, therefore, the invention provides amethod of directing a beam of incident light to a diffusely scatteringsample, comprising:

locating a delivery filter adjacent to the sample, or covering a regionof the sample with the filter, the delivery filter havingcharacteristics such that reflection of said incident light is dependentupon angle of incidence of said incident light at the filter; and

directing a beam of said incident light through the delivery filter at abeam angle of incidence, which may preferably be approximately normalincidence, and to the sample, such that incident light diffuselyscattered back from the sample towards the delivery filter ispreferentially reflected by the filter back towards the sample.

According to another aspect, the invention provides a delivery filterhaving a transmission edge which lies to one side of the wavelength ofthe incident light at approximately normal incidence, for example lessthan 10° from perpendicular, thus permitting an incident beam to pass,and which lies at the other side of the wavelength of the incident lightat shallower angles of incidence, for example greater than 30°, thusreflecting back diffusely scattered light emerging from a sample.

The invention also provides corresponding apparatus. For example, thedelivery filter may be considered as an optical window, or an enclosureor cover, or may be part of a more extensive optical enclosure or coverfor the sample having further optical components. An aspect of theinvention then provides an optical cover for enhancing the intensity ofincident light within a diffusely scattering sample comprising adelivery filter through which a beam of said incident light may bedirected into the sample at a beam angle of incidence with said filter,the delivery filter having characteristics such that reflection of saidincident light increases at angles of incidence away from the beam angleof incidence, such that incident light diffusely scattered out of thesample is preferentially reflected back into the sample by the deliveryfilter.

The delivery filter is preferably positioned adjacent to the sample, tomaximise the return of incident light to the sample, and minimise escapeof backscattered light around the edges of the filter. For example, ifthe filter has a diameter “d” then it may preferably be positionedwithin one diameter distance d from the sample, and more preferably halfa diameter d/2, or more preferably still within a distance of aboutd/10. In practice, it may be preferable to locate the filter as close aspossible to, for example touching, the sample. Typically, the filter maybe parallel or approximately parallel to the underlying sample surface.Light may be collected by transmission back through the delivery filter,for example by using a filter having a transmission region covering thespectral features of interest, as discussed in detail below.Alternatively, a separate collection filter could be used having asuitable transmission region which preferably excludes the wavelength ofthe incident light for a wide range of angles of incidence.

The invention may be used in a variety of applications, such as toprovide an optical enclosure around a pharmaceutical tablet or otherobject to be tested by spectroscopic analysis, or to provide a window toa tissue sample, or to part of a human or animal subject, through whicha beam of incident light is directed.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described, by way of exampleonly, with reference to the accompanying drawings, of which:

FIG. 1 illustrates schematically the illumination by, a incident beam 14of a sample 10 through a delivery filter 30, and collection of scatteredlight for spectral analysis;

FIGS. 2 a and 2 b show transmission characteristics for two exampledelivery filters;

FIG. 3 shows the wavelength of the transmission feature of a narrow bandpass dielectric filter at various angles of incidence, as a ratio withthe wavelength of the transmission window at normal incidence;

FIG. 4 illustrates a more complete optical enclosure extending that ofFIG. 1 to include mirror surfaces 54 and a collection filter 50;

FIG. 5 shows transmission characteristics for an example collectionfilter;

FIG. 6 shows an arrangement in which incident light delivery andcollection use the same filter;

FIGS. 7 a and 7 b show transmission characteristics for two examplecombined delivery/collection filters;

FIG. 8 illustrates the illumination of a sample by an incident beam andthe collection of light at two separate spacings on the same side of thesample.

FIGS. 9 a and 9 b illustrate, in plan view, alternative configurationsfor delivery and collection filters disposed on the same side of asample;

FIGS. 10 a to 10 d illustrate arrangements for applying the invention toa curved sample surface;

FIG. 11 a illustrates geometry of a sample used in a mathematical modelused to demonstrate the invention;

FIGS. 11 b to 11 f illustrate arrangements of a delivery filter 212, acollection filter 214, and mirrored surfaces 216 around the sample ofFIG. 11 a;

FIGS. 12 a and 12 b, 13 a, 13 b, 14 a and 14 b show calculatedintensities of Raman scattered photons emerging from the sample of FIG.11 a according to various configurations of filters and mirroredsurfaces used in the mathematical model, and related enhancementfactors;

FIG. 15 illustrates a geometry of a central delivery filter andsurrounding annular collection region applied to the model sample ofFIG. 11 a;

FIGS. 16 a and 16 b show calculated intensities using the geometry ofFIG. 15, and related enhancement factors;

FIG. 17 schematically shows a laboratory optical arrangement used todemonstrate the invention;

FIGS. 18 a and 18 b show Raman spectra measured using the apparatus ofFIG. 17; and

FIG. 19 illustrates an optical delivery device embodying the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Referring to FIG. 1 there is shown, schematically, apparatus fordetermining characteristics of a diffusely scattering or turbid sample10 using spectroscopy. In this particular example transmission Ramanspectroscopy is used, although other techniques such as infraredabsorption or fluorescence spectroscopy could be used.

A laser 12 forms an incident beam (probe beam) of laser light 14 whichis directed towards the sample 10 by delivery optics 16. The beam entersthe sample, and after scattering within the sample some photons arecollected by collection optics 18. One or more spectral components ofthe collected light are detected by detector 20, such as a spectrometer,and results of the detection may typically be passed to a computer orother analyser device 22 for data storage and/or interpretation. Inparticular, photons which have been inelastically Raman scattered todifferent wavelengths within the diffusely scattering sample may bedetected and analysed.

For many applications, especially in Raman spectroscopy for which thescattering cross sections are small, a high probe beam intensity isdesired to increase the intensity of the spectral components to bedetected. However, this may cause damage to the sample. An alternativeapproach, therefore, is to use a long exposure time, although this maybe impractical for other reasons, for example the time available to makea measurement may be small such as on a production line. Another optionto improve the collected spectral signal is to use a probe beam ofhigher cross sectional area. In the example of FIG. 1 this latter optionhas been used with the delivery optics delivering a wide diameter beamto the sample to increase total beam power without unduly increasingbeam intensity.

A delivery filter 30 is positioned adjacent to, and may even be incontact with, the sample, to form, or partly form an at least partialoptical enclosure 31 of or optical cover over the sample, and the probebeam is directed into the sample through this delivery filter. Thefilter has transmission characteristics which allow the probe beam topass through the filter and into the sample, but which tend to block thereturn of probe light scattered back from the sample. A substantialportion, for example at least 50%, of the probe light scattering out ofthe sample is instead reflected by the delivery filter back into thesample, thereby increasing the intensity of probe light within thesample, and thereby the strength of the spectral components in thecollected light which are to be detected. Preferably this is achievedwith only a minimal reduction in the power of the beam as it initiallypasses through the filter.

The desired characteristics of the delivery filter may be provided by afilter having transmission and/or reflection characteristics which shiftin wavelength depending upon the angle of incidence of light at thefilter, and example characteristics of such a filter are shown in FIG. 2a. The transmission characteristic for photons arriving at the filter atnormal incidence is shown by the solid curve 40, which defines anapproximately Gaussian transmission window. In this example the centreof the transmission window is coincident with the wavelength of theprobe laser light 42, although this is not strictly necessary. Forhigher angles of incidence the transmission window moves to smallerwavelengths, for example as shown by the broken curve 44 for a 90 degreeangle of incidence. As can be seen from the figure, probe lightscattered back out of the sample and returning to the delivery filter atangles of incidence of greater than a few degrees away from normal willbe reflected back into the sample, increasingly for higher angles ofincidence, rather than being transmitted away from the sample throughthe delivery filter. The same effect is achieved by using the shortwavelength transmission edge filter of FIG. 2 b, with the edgewavelength matched to transmit light of the probe beam wavelength at thebeam angle of incidence at the filter, and to reflect light of the sameprobe beam wavelength at larger angles of incidence.

The transmission window of FIG. 2 a may be conveniently termed a probelight (or incident light) transmission feature, which shifts to shorterwavelengths away from the probe wavelength, for increasing angles ofincidence. The low transmission region to the long wavelength side ofthe transmission window may similarly be termed an incident lightreflection feature.

In FIG. 2 b the transmission feature is provided by the long wavelengthend of the transmission window, and the reflection feature by the lowtransmission region immediately beyond the edge.

The desired characteristics of the delivery filter may also be expressedin terms of a transmission edge 41 characteristic positioned to one sideof the incident light wavelength band, close to normal incidence, andlying at the other side of the incident light wavelength at greater thana threshold angle of incidence, which could be 10°, 20° or other anglesaway from perpendicular, depending on the breadth of the incident lightwaveband, the type of filter used, and so on.

For the described mechanism to be effective it is necessary to use aprobe beam which is collimated or at least semi-collimated so as to havea significantly smaller average angle of incidence at the filter thanthe average angle of incidence of beam photons subsequently scatteredback out of the sample towards the delivery filter. Typically, the beamphotons should have an average incidence angle of less than about 10degrees in the presently described examples.

A typical wavelength range of Stokes shifted Raman scattered spectralfeatures to be detected and analysed by the arrangement of FIG. 1 isshown as broken box 46 in FIGS. 2 a and 2 b. It can be seen that thefilter characteristics have the additional benefit of blocking, andpreferably reflecting back into the sample, Raman scattered photons, forsubsequent collection by the collection optics.

A delivery filter having suitable characteristics may be provided by adielectric multilayer filter, otherwise known as a thin-filminterference filter, and in particular a narrow band pass filter of thistype. Suitable filters are manufactured, for example, by Semrock, Inc.,with information available at http://www.semrock.com. A catalogueMaxLine® Laser-Line narrow band pass filter or short wavelength passedge filter could be used. Suitable band pass filters currentlyavailable from Semrock, Inc. have band pass widths from about 1.2 nm to4.0 nm over a corresponding band pass range of close to 325 nm to 1064nm.

The spectral shift of a multilayer dielectric filter as a function ofangle of incidence can be derived from the following formula:

λ=λ_(o)√{square root over (1−(sin Θ/n _(eff))²)}

In this formula, λ_(o) is the wavelength of a spectral feature of thefilter at the normal incidence and λ is the new wavelength of thefeature for photon incidence angle Θ, n_(eff) is the effectiverefractive index which is the refractive index of the filtermedium/ambient refractive index (eg air). The formula inherently impliesthat only blue shifts are possible as the square root term is alwayssmaller or equal to 1. FIG. 3 illustrates the dependence of λ/λ_(o) onthe angle of incidence. The plot assumes a constant value of therefractive index, set to 1.45, which corresponds to the refractive indexof fused silica at ˜800 nm. The ambient medium is assumed to be air.

From the above formula it can be estimated that a 10 degree tilt awayfrom the normal incidence of the impacting photons results in a shift ofthe central wavelength of a band pass filter by about 6 nm. This issufficiently far away from the laser wavelength for a typicalnarrow-band pass filter to result in the reflection, rather thantransmission, of photons emerging from the sample at this or higherangles. Based on a simple geometric consideration, assuming that all ofthe photons impacting at the filter within a 10 degree half angle cone,and those at larger angles are reflected, then only about 1.5% of thephotons emerging isotropically from the sample would be transmittedthrough the filter, and the remaining 98.5% would be reflected back intothe sample. This calculation reflects the very low absorption losses indielectric and similar filters such that the transmission fraction T andreflection fraction R are related as T+R≈1.

A filter may be obtained which matches the particular wavelength of theprobe beam 14, in which case the probe beam can be directed through thedelivery filter at or close to normal incidence. In this way the solidangle of incidence at which light scattered from the sample is reflectedis maximised. However, if the central transmission wavelength of thefilter at normal incidence is lower than the probe beam wavelength, thenthe beam may be directed at the delivery filter at a non-normalincidence to give a better wavelength match. This will tend to increasethe solid angle at which probe light transmission takes place, andreduce effectiveness of the delivery filter in returning scattered lightto the sample.

In FIG. 2 a the characteristics of a narrow band pass filter are shown.However, filters having other characteristics can be used such as aspectrally broader band pass filter having a steep long wavelength edgepositioned to transmit the probe light at only low angles of incidence,a short wavelength transmitting edge filter used in the same way asillustrated in FIG. 2 b, or any other filter having a similar effect.

In FIG. 1 the delivery filter is illustrated as a partial, or part of apartial or complete optical enclosure around the sample. Proximity ofthe delivery filter to the sample is important. To be effective, asignificant proportion of the probe light scattered out of the sampleshould be reflected back into the sample by the delivery filter. Sincein the examples given above the amount of reflection is dependent onangle of incidence, the delivery filter should preferably be no furtheraway from the sample than the diameter of the filter, or the size of thesample, whichever is smaller, and more preferably no further away thanone tenth of this distance. Typically distances of up to a few mm, forexample less than 5 mm, may be appropriate.

FIG. 4 illustrates a further developed optical enclosure disposed aroundthe sample 10 of FIG. 1. Although not illustrated, the laser beam isdelivered, and light collected and analysed in much the same way as inFIG. 1. The principal difference is that further optical elements havebeen added to the enclosure 31 close to the sample 10 in order tofurther increase the intensity of probe light within the sample, and toreduce the loss of those spectral components which are to be detectedand analysed.

Disposed between the sample and the collection optics is an optionalcollection filter 50. This filter is selected to block the majority ofprobe light emerging from the sample towards the collection optics 18,and preferably to reflect at least a substantial portion of this probelight back into the sample. The filter is also selected to allowscattered light of longer wavelengths to pass, in particular photonswhich have been Stokes shifted by Raman scattering.

Suitable transmission characteristics for the collection filter areillustrated in FIG. 5. It will be seen that the transmission curve has asteep edge between the probe wavelength 42 and the wavelengths ofspectral interest 46, so that the filter reflects probe light back intothe sample but allows Raman scattered photons to pass. If a filterhaving characteristics which shift to smaller wavelengths withincreasing angle of incidence is used, then design constraints mayresult in probe light at higher angles of incidence passing through thefilter in significant amounts. A further filter in the collectionoptics, such as a narrow band notch filter, may be used to eliminate anyremaining photons of the original wavelength present in the collectedlight.

A long-wave-pass dielectric multi-layer edge filter may be used for thecollection filter, for example a catalogue RazorEdge® filter currentlymanufactured by Semrock, Inc. Such filters are available with edges at arange of wavelengths from infrared to ultraviolet, with a pass bandwidth of typically 100 nm to 1000 nm, and an edge transition width ofbetween about 100 cm⁻¹ and 500 cm⁻¹. Of course, a filter of especiallyselected characteristics may be used if required.

To be effective in returning probe photons back into the sample, theedge of the collection filter characterised in FIG. 5 must be at asufficiently longer wavelength than the probe wavelength 42 to permitthe filter to reflect probe photons arriving at a wide range of angles,for example in the region of 1000 cm⁻¹ to 2000 cm⁻¹ above the probewavelength. In practice, this may restrict the range of Raman spectralfeatures detectable to wavelengths at least this much longer than theprobe wavelength, for the collection filter to be effective in returningprobe photons into the sample.

The optical enclosure 31 shown in FIG. 4 optically includes or iscompleted with one or more optically reflecting mirror surfaces 54around the sample. There is no need for these surfaces to transmiteither probe light or light to be collected, so elements reflecting atleast at the probe wavelength and at the wavelengths of spectralcomponents to be detected, over a wide range of angles of incidenceshould be used.

To maximise the effect of the cavity, the delivery and collectionfilters may be sized to cover only the areas needed for probe lightdelivery and light collection, with substantially all of the remainingenclosure provided by highly reflective mirrored surfaces.

Some alternative embodiments and configurations using the invention areillustrated using FIGS. 6-9 b. In FIG. 6 the laser beam 14 is deliveredto and light is collected from the same part or surface of the sample10. To achieve this a combined delivery/collection filter 52 is used toboth transmit the probe beam to the sample and to transmit light to beanalysed by detector 20 and analyser 22. The combined filter 52 islocated proximal or adjacent to the sample in the same way as thedelivery filter in the examples of FIGS. 1 and 3 to form at least partof an optical enclosure 31 for the sample. The enclosure may alsopresent reflective or mirrored surfaces 54 to parts of the sample, andmay additionally include a separate collection filter.

The combined filter 52 has characteristics which allow transmission ofspectral features wanted for detection and analysis, while reflecting asignificant portion of light having the wavelength of the probe beamback into the sample. Transmission characteristics of a suitable filterare presented in FIGS. 7 a and 7 b. The filter of FIG. 7 a combines anarrow transmission window 60 coincident with the probe wavelength 42,shown in the figure as having an approximately Gaussian form, with alow-pass edge transmission window 62. In FIG. 7 b a reflecting featureof a notch filter blocks wavelengths lying in a region between the probewavelength 42 and the Raman spectral feature region 46. In both cases,the filter characteristics shift to shorter wavelengths as incidenceangle is increased, as already described above, with the broken curvesshowing the transmission at an angle of 80 degrees. It can be seen thatat all angles the Stokes shifted Raman spectral features in region 46are transmitted through the filter to be collected and analysed. Probewavelength light scattering out of the sample to the combined filter atangles away from normal incidence is reflected back into the sample to alarge extent.

Filters having suitable characteristics similar to those shown in FIGS.7 a and 7 b can be constructed using known thin-film interference filtertechniques. Other types of filter construction and filters having othercharacteristics may also be used to achieve a combineddelivery/collection filter. Such filters can also be used as a deliveryonly filter, although with unwanted loss of the Raman shifted lightthrough the filter.

Although FIGS. 1 and 4 illustrate a transmission geometry where light isdelivered and collected at opposing sides of a sample, and FIG. 6illustrates a reflection geometry in which delivery and collection takeplace in the same or closely spaced regions, a range of other geometriesmay be used. In FIG. 8 a single combined delivery/collection filter 52is used. Delivery optics 16 direct the probe laser beam 14 through afirst region of the filter into the sample, and one or more separatecollection optics 18 collect light transmitted through one or morefurther regions of the filter spaced from the first region. As describedin WO2006/061566, this geometry can be used to determine spectralcharacteristics of the turbid sample from a controlled profile of depth.With a single collection optic the analyser can be arranged to rejectsurface spectral features if known in advance to select for spectralfeatures from depth, while with multiple collection optics adeconvolution of spectral characteristics from different depths can becarried out using assumptions regarding the expected contributions fromdifferent depths depending on distance of collection from the deliveryregion.

In variations to the geometry of FIG. 8, one or more dedicatedcollection filters such as that discussed in connection with FIG. 5 areused between the sample and the collection optics. In FIG. 9 a a centraldelivery filter (d) is surrounded by an annular, concentric collectionfilter (c). In FIG. 9 b a central collection filter (c) is surrounded byan annular concentric delivery filter (d). The collection and deliveryoptics for such arrangements may conveniently comprise a bundle ofoptical fibres, for example as discussed in WO2006/061566. Clearly,various other geometries could be used. For example, either the deliveryor the collection filter could be omitted, and various continuous andsegmented shapes could be used for the regions.

Optical elements suitable for use as the described delivery filter,especially dielectric multilayer filters, are readily availablecommercially as flat elements. As described above, it is desirable forthe filter to be located close to the surface of the sample, to maximisereflection by the filter of scattered incident light back towards thesample. Clearly, if the surface of the sample in the region of thedelivery filter is strongly curved instead of flat, the effect of theinvention may be reduced. FIGS. 10 a to 10 d illustrate ways in whichthe otherwise adverse effects of a curved sample surface may bemitigated.

FIG. 10 a shows, in cross section, a diffusively scattering sample 104having a curved surface. An example of such a sample might be apharmaceutical capsule or tablet. As in previous figures, a laser 100forms an incident beam of laser light 102 which is directed towards thesample 104 by delivery optics 106. Collection optics, detector andanalysis elements are not shown in FIG. 10 a, but of course may bepresent as required. The incident beam enters the sample 104 through adelivery filter 108 which is itself curved so as to match, at leastapproximately, the surface of the underlying sample 104. The deliveryfilter has optical characteristics as described above, including thecharacteristic of preferentially allowing the incident beam to pass atangles close to normal to the filter, while reflecting back to thesample diffusely scattered light of the same wavelength emerging fromthe sample at a wider range of angles, for example beyond around 10°from perpendicular.

To provide optimal transmission of the incident beam through the curveddelivery filter, the delivery optics 106 are adapted to form the beamsuch that the angle of incidence is close to normal across the surfaceof the filter. For a convex sample surface and delivery filter thismight be achieved by an appropriate concave lens or suitably shapedmirror.

Because curved dielectric filters are likely to be expensive and moredifficult to obtain or manufacture than flat filters, it would bedesirable to adapt the invention for use on curved sample surfaces whilestill using a flat filter. In FIG. 10 b this is achieved by disposing adiffusely scattering spacer element 110 between a sample surface and aflat delivery filter 112. The spacer element 110 may comprise, forexample, an elastomer such as a silicon polymer containing isotropicscattering centres such as micrometer sized particles. The spacerelement 110 may be rigid or semi rigid, and shaped to conform to theunderlying curvature of the sample 104. Alternatively, the spacerelement may be sufficiently flexible to conform to the sample 104, froma relaxed shape which is either an approximation to the sample, or adifferent shape such as a flat surface. An advantage of using anelastically deformable spacer element is that it may conform closely tothe sample surface against which is pressed. An advantage of thisarrangement is that the delivery optics 114 do not need to supply anappropriately converging or diverging beam, since only a collimatedincident beam is required.

A variation on the described arrangement of FIG. 10 b is to include inthe spacer element 110 anisotropic scattering characteristics, favouringthe sample to filter scattering direction. This can be achieved, forexample, by including fibre elements, such as silica fibres, extendingin this direction, mixed with diffusely scattering sphericalmicrometer-size particles.

Another arrangement for adapting a flat delivery filter 112 to a curvedsample 104, using peripheral mirrored guiding surfaces 116 is shown inFIG. 10 c. The surface of the sample 104 to be covered by the deliveryfilter 112 is convex, and the resulting gap between the filter and thesample is provided with the peripheral mirrored guiding surfaces, so asto prevent or reduce the escape of diffusely scattered light emergingfrom the thereby enclosed sample surface, and to channel this light tothe delivery filter, for reflection back towards the sample 104.

Typically, the peripheral mirrored guiding surfaces will beapproximately perpendicular to the delivery filter, and extend around acircumference of the space between the filter and the curved surface ofthe sample to be covered. Such a peripheral mirrored guide isadvantageous in providing improved coupling, without deformation oradaption, to a range of sample surface curvatures including flatsurfaces.

FIG. 10 d illustrates an arrangement combining the approaches of FIGS.10 b and 10 c, with the described spacer element 100 being provided withthe described mirrored guiding surfaces 116 about its periphery.

Numerical Model

A numerical model already described in Matousek, P. et al., AppliedSpectroscopy 59, 1485 (2005) was used to demonstrate the effectivenessof the optical enclosure 31 described above. Briefly, both elasticallyscattered probe beam photons and non-elastically scattered (eg Ramanscattered) photons are individually followed as they propagate through amodelled medium in random walk-like fashion in three-dimensional space.A simplified assumption is made that in each step a photon propagates ina straight line over a distance t and thereafter its direction is fullyrandomised at the next scattering event. This is somewhat simplisticfrom the standpoint of individual scattering events which are oftenstrongly biased towards the forward direction. However, for largenumbers of scattering events, as of interest here, this simplificationis justifiable with an appropriately chosen randomisation length. Thepropagation distance, t, over which the photon direction is randomised,can be crudely approximated as the transport length of the scatteringmedium l_(t), which is defined as the average distance photons musttravel within the sample before deviating significantly from theiroriginal direction of propagation.

As shown in FIG. 11 a, the model considers the sample 200 to be ahomogeneous turbid medium having the shape of a short cylinder with aradius of 6 mm. A first air-medium interface 202 is located at a topcircular surface with z=0, where z is a Cartesian coordinate normal tothe interface plane. The other sample-to-air interfaces exist at theopposite circular surface 204 of the sample at a position z=d, where dis the sample thickness, and on the cylindrical side wall 206 of thesample. The thickness of the sample d was varied between simulations,from 0.5 mm to 6 mm in 0.5 mm steps.

The model assumes that all the probe photons are first placed at a depthequal to the transport length it and symmetrically distributed aroundthe origin of the co-ordinate system x,y. The radius of the probe beam208 of incident light is r=3 mm and the beam has uniform intensity, witha flat, ‘top-hat’ intensity profile with all the photons having equalprobability of being injected into the sample at any point within thebeam cross-section.

The numerical code was written in Mathematica 5.0 (Wolfram Research).100,000 photons were propagated separately, each across an overalldistance of 400 mm (2000 steps) which is in line with observed migrationtimes in Raman spectroscopy. If not detected or lost from the mediumwithin this propagation distance, the photons were assumed to beabsorbed by the medium itself which might be the case in the presence ofvery weak absorption (OD ˜1 per 40 cm).

The optical density accounting for the conversion of probe photons intoRaman photons was set to 1 per 1000 mm. Although this value is higherthan that of real conversion, it only affects the absolute number ofRaman photons, and not the spatial dependencies of concern to asignificant degree in the studied regime and was verified by varyingthis value up and down. The step size used was t=0.2 mm. Thiscorresponds to powder particle sizes of 10 and 20 μm diameter for ananisotropy of 0.9 and 0.95, respectively. The calculations were repeated10 times summing all the detected Raman photons in these repeated runs.

The model assumes two different collection geometries. In a firstgeometry, light is collected at the top sample surface from the sameregion 210 on the sample surface as the probe beam entry (backscatteringgeometry). In a second geometry, light is collected from the oppositesurface of the sample from a congruent region 211 centred around theprojection axis of the probe beam (transmission geometry). The modelcalculations were first performed for both the transmission andbackscattering geometries assuming no filters or reflective elements.

The transmission geometry calculation was then carried out with thepresence of particular optical enclosure elements as illustrated inFIGS. 11 b-11 f. In FIG. 11 b a delivery filter 212 is provided by aband pass filter transmitting all the probe photons from above andreflecting 95% of the probe and Raman photons from below. The deliveryfilter is positioned adjacent to the upper surface 202 of the sample,covering the whole surface. In FIG. 11 c a collection filter 214 isprovided by an edge filter transmitting all the Raman photons andreflecting 95% of the laser photons back into the turbid medium. Thecollection filter is positioned at the lower surface 204 of the sample,and there is no delivery filter at the upper surface.

In FIG. 11 d the delivery filter 212 is provided, and the sidewall 206of the sample is surrounded by a 100% reflective enclosure 216 to returnboth probe wavelength and Raman scattered photons into the sample, butno collection filter is used. In FIG. 11 e, the delivery filter 212 andcollection filter 214 are present, and in FIG. 11 e all threecomponents, including the sidewall mirrored reflective element, arepresent.

Results of the Monte Carlo simulations for the various transmissiongeometries of FIGS. 11 b-11 f are shown in FIGS. 12 a and 13 a, in whichthe ordinates represent counts of Raman scattered photons collected atthe collection region 210 or 211, and the abscissae represent differentthicknesses of sample. In FIG. 12 a curve 220 results from thetransmission geometry being used with no optical enclosure elements.Curve 222 results from the backscattering geometry being used, againwith no enclosure elements.

The signal in the backscattering geometry is about 3 times higher thanthat for the transmission mode for a bare 4 mm thick sample, which is atypical thickness for a pharmaceutical tablet. The signal for thebackscattering mode rises monotonically with increasing samplethickness, a behaviour observed experimentally previously. For thetransmission geometry the signal intensity initially increases with thetablet thickness due to larger photons pathways available for theconversion of photons into Raman photons, but beyond about 3 mm thesignal starts diminishing, an effect ascribed to increased lateralphoton transport causing more photons to miss the collection aperture.

Curve 224 results from the transmission geometry being used with thearrangement illustrated in FIG. 11 b, with the probe beam passing intothe sample through delivery filter 212. The model predicts anenhancement of the transmission Raman signal collected at the oppositeface by a factor of about 9.4 for a 4 mm thick sample. Interestingly,this signal level considerably exceeds even that of the backscatteringRaman signal for the unenclosed sample. The ratio of the transmissiongeometry curves with and without the delivery filter (224, 222) isplotted as an “enhancement factor” 224′ in FIG. 12 b, which lies betweenabout 8 and 10 across most of the thickness range above 1 mm.

In FIG. 13 a, curve 220 again represents Raman scattered photonscollected in a transmission geometry with no optical enclosure elements.Curve 230 results from the addition of collection filter 214, but nodelivery filter 212, as illustrated in FIG. 11 c. As shown in thecorresponding enhancement factor curve 230′ in FIG. 13 b, the collectionfilter on its own gives rise to about a doubling of the detected Ramanphotons. This is a much weaker effect than use of the delivery filteralone, as expected because the largest photon loss in absence of anyenclosure element is at the point of entry of the probe beam.

The delivery filter curve 224 of FIG. 12 a is shown for comparison, andthe modest increase in detected Raman intensity achieved by using theadditional mirror sidewall mirror element as illustrated in FIG. 11 d isshown as curve 234 (and as enhancement factor curve 234′ in FIG. 13 b).For sample objects 200 of reduced diameter or increased thickness thesidewall mirror enclosure would provide larger enhancements.

Curve 238 is for the configuration of FIG. 11 e, where the delivery andcollection filters are used but without the mirrored sidewall element.The enhancement factor 238′plotted in FIG. 13 b, is about 27.5 for theminimum 0.5 mm thick sample, falling continuously to about 14.6 at 4 mm,and down to about 10, still higher than the curve 224′, for deliveryfilter only for 6 mm thickness.

Finally, curve 242 illustrates the case of the arrangement of FIG. 11 f,in which all three of the delivery filter, collection filter, andsidewall mirror elements are used, with an enhancement factor 242′falling more slowly than curve 238′ as the sample thickness increases,to a factor of about 12 at 6 mm thickness.

Overall, for a thicker sample, the single most beneficial enclosureelement is the delivery filter. For thinner samples, the additionalbenefits of using the collection filter are very significant, but reducewith increased sample thickness as the proportion of probe photonsreaching the far side of the sample diminishes.

Results of Monte Carlo simulations for the backscattering geometry areshown in FIG. 14 a. Curve 222, representing the count of Raman photonsemerging from the upper surface of the sample using no optical enclosureelements, has already been shown in FIG. 12 a. Curve 248 is for the samebackscattering geometry, but with a combined delivery/collection filterplaced against the top surface of the sample. This arrangement istherefore the same as shown in FIG. 11 a, except that the filter isdefined to allow all Raman scattered photons to exit, while returning95% of the probe photons back into the sample. Filter characteristicswhich could be used to achieve this or similar performance are shown inFIGS. 7 a and 7 b. For a 4 mm thick sample, the enhancement factor overthe bare sample results of curve 222 is about 5.6, as illustrated in theenhancement factor curve 248′ of FIG. 14 b.

The compromises of using an filter having an edge characteristic to passRaman scattered photons while blocking the majority of scattered probephotons were mentioned in the discussion of FIG. 5 above. Essentially,the edge must be far enough beyond the probe wavelength to reflectscattered probe photons incident at shallow angles, without blockingdesired Raman wavelength photons. The enhancement factor for a 4 mmthick sample has been calculated for a probe wavelength of 830 nm and adielectric filter having the frequency dependence of FIG. 3 for an edgecharacteristic lying (for normal incidence) between the probe wavelengthand the Raman wavelength. For an edge lying at each of 1000, 2000 and3000 cm⁻¹ above the probe wavelength, the enhancement factor iscalculated to be 1.8, 2.8 and 4.3.

In a further Monte Carlo experiment the sample arrangement of FIG. 15was used. The cylindrical sample 206 is the same as that of FIG. 11 a. Adelivery filter 260 is placed adjacent to the upper surface, but coversonly a central, circular interface region which has a diameter of 4 mm,centred on a probe beam deposition region 262 with a diameter of 1 mm.The delivery filter 260 is characterised by reflecting back into thesample 95% of the probe and Raman scattered photons which wouldotherwise escape. Raman scattered photons emerging from the uppersurface of the sample in an annular collection region 264 having innerand outer diameters of 6 mm and 8 mm, centred on the probe beamdeposition region 262, were counted.

The counts of Raman photons emerging through the annular collectionregion 264 are shown for a variety of thicknesses of the sample 200 inFIG. 16 a, as curve 266. The counts for a corresponding experiment butomitting the delivery filter 260 are shown as curve 268. The ratio ofthese curves is shown as an enhancement factor, showing the benefit ofusing the delivery filter for a variety of sample thicknesses, in FIG.16 b (curve 270). For a 4 mm thick sample the enhancement factor is 6.7.

The configuration of FIG. 15 is similar to that already discussed inconnection with FIG. 9 a. In one alternative to the configuration ofFIG. 15, an annular delivery filter and deposition region may surround acentral collection region. A variety of other delivery and collectiongeometries to which the delivery and collection filter elementsdescribed herein can be applied are discussed, for example, inWO2006/061566, the content of which is incorporated herein by reference.

Laboratory experiments were also carried out to demonstrate theinvention, using apparatus illustrated schematically in FIG. 17. A probebeam 304 was generated using an attenuated 115 mW temperature stabiliseddiode laser 300 for Raman spectroscopy operating at 827 nm (micro LaserSystems, Inc, L4 830S-115-TE). The beam was spectrally purified byremoving any residual amplified spontaneous emission components from itsspectrum using two 830 nm bandpass filters 302 (Semrock). These wereslightly tilted to optimise their throughput for the 827 nm laserwavelength. The sample 306 was provided by a standard paracetamol tablethaving a diameter of 12.8 mm and a thickness of 3.8 mm, arranged suchthat the probe beam was perpendicularly incident at the centre of acircular face of the tablet, after passing through an adjacent deliveryfilter. The laser power at the sample was 50 mW and the laser spotdiameter was ˜4 mm. The beam was polarised horizontally at the sample.

Raman light was collected from the opposite side of the sample using a50 mm diameter lens 310 with a focal length of 60 mm. The scatteredlight was collimated and passed through a 50 mm diameter holographicnotch filter 312 (830 nm, Kaiser Optical Systems, Inc) to suppress theelastically scattered component of light. The filter was also slightlytilted to optimise the suppression at 827 nm. A second lens 314,identical to the first one, was used to image, with magnification 1:1,the sample collection zone onto the front face of a fibre probe 320 madeof 22 active optical fibres. The individual fibres were made of silicawith a core diameter of 220 μm, a doped silica cladding diameter of 240μm and a polyimide coating of 265 μm diameter. The fibre numericalaperture was 0.37. The bundle was custom made by CeramOptec Industries,Inc. The fibre bundle length was about 2 m and at the output end thefibres were arranged into a linear shape oriented vertically and placedin the input image plane of a Kaiser Optical Technologies Holospec 1.8iNIR spectrograph 322. The Raman spectra were collected using a NIRback-illuminated deep-depletion TE cooled CCD camera 324 (AndorTechnology, DU420A-BR-DD, 1024×256 pixels) by binning the entire chipvertically. The spectra were not corrected for the variation ofdetection system sensitivity across the spectral range.

The delivery filter 308 placed over the laser beam deposition area onthe sample was a 25 mm diameter Semrock dielectric bandpass filtercentred at 830 nm with bandwidth of 3.2 nm (LL01-830-25, MaxLineLaser-line Filter). The slight mismatch between the laser wavelength(827 nm) and the filter wavelength was compensated by introducing asmall tilt to the incident beam at sample 306. Although the mismatchsomewhat reduced the effectiveness of the delivery filter element asubstantial enhancement of the Raman signal was still present.

Raw photon count data from the CCD camera 324 using the abovearrangement is plotted over a range of wavelength difference from thelaser frequency in FIG. 18 a. The lower curve 350 is for the experimentwith delivery filter 308 omitted, and the upper curve 352 is for whenthe delivery filter was in place, in each case for the same exposuretime of 10 seconds. In FIG. 18 b the same data is shown, but with thevertical scale of curve 350 expanded by a factor of 6.5. It can be seenthat a uniform enhancement factor of about 6.5 is achieved by theadditional use of the delivery filter across the whole spectral range.

The experimental enhancement is less than the value of 9.4 found for thecorresponding numerical Monte Carlo experiment, but this may easily beaccounted for in differences in scattering lengths between the modelledand real samples, as well as the slight mismatch between the laser anddelivery filter wavelengths. Nevertheless, the enhancement factor isstill very high.

Importantly, the enhancement exhibited good reproducibility uponsubsequent remounting of the delivery filter adjacent to the tablet, andno temporal fluctuation was observed when the filter was in place. Also,the enhancement was uniform across the Raman spectra measured, which maybe important in applications involving complex analytes where thespectral pattern serves as a means of identifying multiple individualcomponents, as well as determining relative concentrations.

Although the invention has generally been illustrated with embodimentsin which Raman spectroscopy of a sample is required, it may moregenerally be applied to any circumstances in which retention of incidentlight within a scattering medium is required. In some embodiments, forexample, no collection or analysis of the scattered light is required.

FIG. 19 illustrates an optical head 380 for delivering light generatedusing laser 382 into a human or animal patient 384, for example totrigger a chemical reaction involving a substance introduced into thepatient, as known in various photodynamic therapies. The use of adelivery filter 386 adjacent to the surface of the patient, inconjunction with collimator 388′ preferentially retains the introducedlight within the patient, allowing very much lower laser powers to beused.

A particular application is in photo-thermal therapies such asphoto-thermal cancer therapies, in which electromagnetic radiation isdelivered to tissue containing absorbing bodies. In recent research,near-infrared radiation is delivered to tissues containing appropriatelyformed nanoparticles, for example see Gobin et al., Nano Lett., 7(7),1929-1934, 2007. The present invention provides an improved method ofcarrying out photo-thermal therapy by directing the radiation, typicallylaser radiation, into tissue through a delivery filter as describedherein, thereby increasing the intensity of the radiation within thetissue without needing to increase the power of the incident beam.

Other applications include NIR absorption or fluorescence opticaltomography and spectroscopy.

A diffusely scatting sample may be defined, for example, as a samplewithin which the typical path length between scattering events of aphoton of the relevant incident light is much less than the size of thesample, for example at least ten times, and more preferably at least ahundred times less than a characteristic size of the sample (such as thethickness in the axis of the incident light beam), such that thedirectional structure of an incident light beam is very quickly lost.

A variety of changes and modifications may be made to the describedembodiments without departing from the scope of the invention as definedby the appended claims.

1. A method of directing a beam of incident light into a diffuselyscattering sample, comprising: locating a delivery filter adjacent tothe sample, the delivery filter having characteristics such thatreflection of said incident light is dependent upon angle of incidenceof said light at the filter; and directing a beam of said incident lightthrough the delivery filter at a beam angle of incidence and to thesample, such that incident light diffusely scattered back out of thesample to arrive at the filter is preferentially reflected by thedelivery filter back towards the sample.
 2. The method of claim 1wherein the incident light is of a predetermined wavelength, and thedelivery filter is adapted to reflect light of said predeterminedwavelength more strongly when incident at a shallower angles ofincidence.
 3. The method of claim 1 wherein reflection of said incidentlight by the delivery filter increases at angles of incidence higherthan said beam angle.
 4. The method of claim 1 wherein the filtercharacteristics have an incident light transmission feature, which iscoincident with the wavelength of the incident light at the beam angleof incidence, and which shifts to shorter wavelengths for increasingangles of incidence.
 5. The method of claim 1 wherein the filtercharacteristics have an incident light reflection feature at longerwavelengths than the wavelength of the incident beam at the beam angleof incidence, and which shifts to shorter wavelengths to cover the beamwavelength at higher angles of incidence.
 6. The method of claim 1wherein the beam at the delivery filter is collimated orsemi-collimated.
 7. The method of claim 1 wherein the delivery filter isa multi-layer dielectric filter.
 8. The method of claim 1 wherein thedelivery filter is a holographic filter.
 9. The method of claim 1wherein the delivery filter is spaced from the sample by a distancewhich is less than a diameter of the incident beam at the sample. 10.The method of claim 1 wherein the delivery filter is spaced from thesample by a distance which is less than a diameter of the deliveryfilter.
 11. The method of claim 1 wherein the delivery filter is spacedfrom the sample by a distance which is less than a diameter of thesample.
 12. The method of claim 1 wherein the delivery filter is curvedso as to be adapted to conform to a curved surface of the sample coveredby the filter.
 13. The method of claim 1 further comprising providing adiffusely scattering spacer element between a surface of the sample tobe covered, and the delivery filter.
 14. The method of claim 13 whereinthe spacer element is deformable so as to adapt to a curved surface ofthe sample.
 15. The method of claim 13 wherein the spacer element isprovided with anisotropic scattering characteristics.
 16. The method ofclaim 1 further comprising providing a peripheral mirrored guidesurrounding a space between the delivery filter and the surface of thesample covered, so as to retain diffusely scattered light which wouldotherwise escape around the edge of the covered area.
 17. The method ofclaim 1 further comprising collecting light scattered out of the sample,and analysing said light to detect one or more spectral features of saidcollected light.
 18. The method of claim 17 wherein said one or morespectral features are Raman scattering features.
 19. The method of claim17 wherein the collected light is collected after scattering away fromthe sample and through the delivery filter.
 20. The method of claim 17wherein the collected light is collected after scattering away from thesample and through a collection filter separate from said deliveryfilter.
 21. An optical enclosure for enhancing the intensity of incidentlight within a diffusely scattering sample comprising a delivery filterthrough which a beam of said incident light is directed to the sample ata beam angle of incidence with respect to said filter, the deliveryfilter having characteristics such that reflection of said incidentlight increases at angles of incidence away from the beam angle ofincidence, such that incident light scattered diffusely from the sampleis preferentially reflected back into the sample by the delivery filter.22. The optical enclosure of claim 21 wherein the characteristics of thedelivery filter are such that the reflection of said incident lightincreases at increasing angles of incidence.
 23. The optical enclosureof claim 21 wherein the characteristics of said delivery filter includea transmission region which shifts to shorter wavelengths than theincident light for higher angles of incidence.
 24. The optical enclosureof claim 21 wherein the delivery filter has a shorter wavelengthtransmission edge feature, the wavelength of said edge feature shiftingto shorter wavelengths with increasing angles of incidence, such thatthe incident light is transmitted at the beam angle of incidence, andsignificantly reflected at a range of higher angles of incidence. 25.The optical enclosure of claim 21 wherein the delivery filter is a bandpass filter having a band pass wavelength region matched to thewavelength and beam angle of incidence of said beam of incident light.26. The optical enclosure of claim 21 wherein the delivery filter is anotch filter having a blocking wavelength region matched to block theincident light at a range of beam angles greater than the angle ofincidence of said beam of incident light.
 27. The optical enclosure ofclaim 21 wherein said delivery filter is adjacent to said sample. 28.The optical enclosure of claim 27 wherein the delivery filter is spacedfrom the sample by a distance which is less than a diameter of thesample.
 29. The optical enclosure of claim 21 wherein the deliveryfilter is curved so as to conform to a curved sample surface to becovered by the filter.
 30. The optical enclosure of claim 21 furthercomprising a diffusely scattering spacer element arranged between thedelivery filter and a curved surface of the sample.
 31. The opticalenclosure of claim 30 wherein the spacer element is deformable so as toadapt to a curved surface of the sample.
 32. The optical enclosure ofclaim 30 wherein the spacer element is provided with anisotropicscattering characteristics.
 33. The optical enclosure of claim 21further comprising a peripheral mirrored guide surrounding a spacebetween the delivery filter and the surface of the sample to be covered,so as to retain diffusely scattered light which would otherwise be lostbetween the sample and the filter.
 34. The optical enclosure of claim 21arranged such that said delivery filter reflects back towards the sampleat least 50% of the incident light scattered out of the sample andreaching the filter.
 35. The optical enclosure of claim 21 wherein thedelivery filter is a dielectric multilayer filter or a holographicfilter.
 36. The optical enclosure of claim 21 further comprising acollection filter which reflects incident light scattering out of thesample back towards the sample, but transmits spectral features having adifferent wavelength to said incident light.
 37. The optical enclosureof claim 36 wherein the spectral features to be detected are Ramanspectral features Stokes shifted to longer wavelengths than the incidentlight by the diffusely scattering sample.
 38. The optical enclosure ofclaim 36 wherein the collection filter comprises a long wavelength passfilter having an edge lying between the wavelength of the incident lightand the spectral features to be detected.
 39. The optical enclosure ofclaim 21 wherein the characteristics of the delivery filter furthercomprises a transmission feature matched to transmit the spectralfeatures to be detected.
 40. The optical enclosure of claim 21 furthercomprising one or more mirror surfaces arranged across parts of thesample not covered by the delivery filter, to reflect light scatteringout of the sample back into the sample.
 41. Apparatus comprising theoptical enclosure of claim 21, the apparatus being for detectingspectral features of a diffusely scattering sample, the apparatusfurther comprising: an incident light source adapted to form saidincident light beam; delivery optics arranged to direct said incidentlight beam through said delivery filter and to said sample; collectionoptics arranged to collect light scattered from said sample; and adetector arranged to detect one or more spectral characteristics of saidcollected light.
 42. Apparatus for detecting one or more spectralfeatures from a diffusely scattering sample, comprising a deliveryfilter adapted to allow an incident light beam to pass through at anincident beam angle of incidence to reach the sample, characterised inthat the delivery filter is a multi-layer dielectric filter having atransmission characteristic coincident with the wavelength of theincident beam at said angle of incidence, but which shifts to shorterwavelengths at higher angles of incidence such that the filterpreferentially reflects back towards the sample incident light diffuselyscattered from the sample.
 43. The apparatus of claim 42 arranged suchthat at least 50% of the incident light back scattered from the sampleto reach the delivery filter is reflected back towards the sample. 44.The apparatus of claim 42 wherein the delivery filter is positionedwithin a distance from the sample which is less than a diameter of thefilter.
 45. The apparatus of claim 42 wherein the incident beam has abeam diameter, and the delivery filter is positioned within a distancefrom the sample which is less than the incident beam diameter.
 46. Theapparatus of claim 42 wherein the delivery filter is arranged to cover aregion of the sample.
 47. A method of illuminating a diffuselyscattering sample, comprising: covering a region of the sample with adelivery filter; and directing a beam of collimated light of apredetermined wavelength through the delivery filter and into saidsample, wherein said delivery filter is adapted to preferentiallyreflect back to the sample light of said predetermined wavelengthdiffusively scattered out of the sample in said region.
 48. The methodof claim 47 wherein the delivery filter has transmission and/orreflection characteristics which shift to shorter wavelengths atshallower angles of incidence.
 49. The method of claim 47 wherein thedelivery filter characteristics have a transmission region coincidentwith the predetermined wavelength at a first range of angles ofincidence, and is shifted away from the predetermined wavelength at asecond range of angles of incidence.
 50. The method of claim 47 whereinthe delivery filter characteristics have a transmission regioncoincident with the predetermined wavelength at substantially normalangles of incidence.
 51. A method of collecting spectrally changed lightfrom a diffusely scattering sample illuminated with light of an incidentwavelength, comprising: covering a region of the sample with acollection filter; and collecting said spectrally changed light throughthe collection filter, wherein said collection filter is adapted toreflect back to the sample light of said incident wavelength diffusivelyscattered out of the sample in said region, and to preferentially allowsaid spectrally changed light to pass.